Biosensor Using Whispering Gallery Modes in Microspheres

ABSTRACT

A biosensor for detecting the presence of a target analyte is disclosed. The biosensor is formed from microspheroidal particles which have had a binding partner for the target analyte immobilized on their surfaces. The binding partners may be nucleotides; peptides, proteins, enzymes, antibodies and so on. When the analyte binds to its partner, the whispering gallery mode (WGM) profiles of the microspheroidal particles change such that the profile peaks undergo a red-or blue-shift. The immobilised binding partners may include fluorophores and the like so that they emit fluorescence, phosphorescence, incandescence and the like. These fluorophores may take the form of a nanocrystal or quantum dot.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of analytedetection. More particularly, the present invention relates to the useof changes in the Whispering Gallery Mode (WGM) profiles ofmicrospheroidal particles induced by the analyte binding to animmobilized binding partner on the particle to thereby detect thepresence of the analyte. The present invention further relates tomultiplexing protocols and to analytes detected by the WGM profilechanges.

2. Description of the Prior Art

Bibliographic details of references provided in the subjectspecification are listed at the end of the specification.

Reference to any prior art in this specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the common general knowledge in any country.

Rapid advances in genomics and proteomics has highlighted theinadequancies of traditional labor intensive methods for examining anddetecting the interaction between two molecules. There is a necessity todevise extremely sensitive methods for the analysis of the interactionbetween different molecules and the detection of analytes in a sample.The development of methods which allow such analysis without the need tolabel one or other or both analytes and/or binding partners thereof,would be particularly desirable.

At present, gene-chips provide a means for high throughput nucleic acidanalysis using oligonucleotide arrays immobilized to slides. However,this technology is dependent on the labeling of at least one of themolecules.

Attempts to develop methods and devices for examining the interaction ofmolecules which are not dependent on the labeling of one or more of themolecules include biosensors, which are most frequently used forexamining DNA and/or proteins based on optical methods. See, forexample, Baird and Myszka, J Mol Recognit 14:261-268, 2001, Rich andMyszka, J Mol Recognit 15:352-376, 2002, Li et al. Science 299:840-843,2003 and Lin et al. Science 278:840-843, 1997 which describe opticalmethods including interferometric devices. Malmqvist, Nature361:186-187, 1993 discloses surface plasmon resonance sensors (SPR). SPRdetects a limit of <10 pg.mm⁻² mass loading (Karlsson and Stahlberg,Anal Biochem 228:274-280, 1995) and allows real-time detection ofbiomolecular interactions. However, SPR requires specific and expensiveinstrumentation and the ability for multiplexed measurements is limited.

The present invention provides reagents and methods for, inter alia, thedetection of interactions between analytes and their binding partnerswithout need for labeling of either molecule.

SUMMARY OF THE INVENTION

The present invention provides methods and reagents for, inter alia,detecting molecules in a sample. These molecules are referred to hereinas analytes. The presence or structure of the analytes need not be knownand hence the subject method is ideal for detecting hitherto unknownbinding partners of orphan receptors and potential modulators of nucleicacid expression or protein including enzyme activity, folding,antigenicity or function. The methods of the present invention arepredicated, in part, on the phenomenon that optically detectable labelsembedded within or onto a microspheroidal particle will display adistinctive Whispering Gallery Mode (WGM) profile. Reference to“optically detectable” includes reference to detection by spectrometricmeans. When a target analyte interacts with a binding partnerimmobilized to the microspheroidal particle, the WGM profile changesenabling very sensitive detection of even rare binding events.

WGMs allow only certain wavelengths of light to be emitted from theparticle. The result of this phenomenon is that the usual broad emission(10-100 nm wide) bands from, for example, a fluorophore becomeconstrained and appear as a series of sharp peaks correspondingeffectively to standing mode patterns of light within the particle. Inaccordance with the present invention, it has been determined that theWGM profile is extremely sensitive to changes at the surface of themicrospheroidal particle and that the WGM profile changes when themicrospheroidal particle interacts with analytes or molecules within itsenvironment.

Accordingly, one aspect of the present invention contemplates a methodof detecting an analyte, said method comprising contacting at least oneset of microspheroidal particles with a sample putatively comprisingsaid analyte, wherein each particle within a set of microspheroidalparticles comprises an optically detectable label and an immobilizedputative binding partner of said analyte wherein each particle set has adefined Whispering Gallery Mode (WGM) profile, wherein binding of saidanalyte to said immobilized binding partner results in a change in saidWGM profile of said at least one set of microspheroidal particles whichis indicative of the presence of said analyte.

The methods of the present invention may be applied to detect modulationin the WGM profile of a microspheroidal particle wherein said modulationresults from detection of binding or other association of molecules in asample to potential binding particles immobilized to the surface of themicrospheroidal particle. Detection of binding reactions between ananalyte and its binding partner based on sensitive changes in WGMprofiles enables the identification and isolation of the analytes.

A feature of the present invention is that the microspheroidal particlesmay be excited with a wide range of light sources, facilitatingmeasurement in many different WGM profiles.

An “optically detectable label” may be any molecule, atom or ion whichemits fluorescence, phosphorescence and/or incandescence. In onepreferred embodiment of the present invention, the optically detectablelabel is a fluorophore, which may encompass a range of opticallydetectable labels such as chemical fluorophores and dyes as well asquantum dots.

The present invention also provides a microspheroidal particle asdescribed herein immobilized to a solid support, for example, a solidsupport may include a microscope slide.

In one specific embodiment, the present invention provides amicrospheroidal particle comprising a latex or silica particle which is1 μm to 100 μm in diameter, labeled with an optically detectable label,such as a fluorophore or quantum dot, the particle further comprising aputative binding partner of an analyte to be detected. The opticallydetectable label is detectable at visible wavelengths and themicrospheroidal particle exhibits one or more WGM profiles. One or moreof the WGM profiles of the microspheroidal particle detectably modulateswhen analytes interacts with the immobilized binding partner on theparticle. Any such change in WGM profile is indicative of the presenceof an analyte which has bound to its binding partner.

Examples of analytes and binding partners include chemical molecules,nucleic acid molecules, proteins, lipids, fatty acids and carbohydrates.

The subject method of the present invention does not require anyknowledge of the existence, presence or structure of the analyte. Forexample, if it is desired to find a molecule (i.e. an analyte) whichinteracts with an enzyme or the catalytic site of an enzyme or at ornear an antigenic epitope of an enzyme or protein, then the subjectmethod does not require knowledge of whether such an analyte exists. Inthat case, the microspheroidal particle will carry the target bindingpartner at or near its surface and changes in WGM profiles used todetect an analyte in a sample such as a combinational library, chemicallibrary or natural product source (e.g. environmental sample, serum,plasma or biological extract) which interacts with the target bindingpartner.

The present invention enables multiplexing by incorporating multiplesets of microspheroidal particles wherein each set comprises particlesof different sizes and/or labeled with different fluorochromes and/ordifferent target binding partners.

The present invention further provides analytes identified by thesubject method and kits useful for practicing the subject method.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

A list of abbreviations used herein is provided in Table 1.

TABLE 1 Abbreviations Abbreviation Description APS Aminopropylsilane GAGGlycosaminoglycan HEX hexachlorofluorescein HLGAG Heparin-LikeGlycosaminoglycan IR Infrared JOE 7′-dimethoxyfluorescein NIR NearInfrared PEI Polyethylene imine PSS Polystyrene sulfonate PVP Polyvinylpyrrolidone QD Quantum Dot SPR Surface Plasmon Resonance TAMRACarboxytetramethylrhodamine TET Tetrachlorofluorescein TIR TotalInternal Reflection UV Ultraviolet WGM Whispering Gallery Mode

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graphical representation showing a schematic of theprocedure used to produce Quantum Dot labeled microspheroidal particles.

FIG. 2 is a graphical representation showing a calculated curve showinghow small changes to the radius of a silica microspheroidal particlelead to clear changes in the calculated WGM profile of themicrospheroidal particle.

FIG. 3 is a graphical representation showing microspheroidal particlescomprising Quantum Dot (QD) optically detectable labels, which exhibitclear distinctive fluorescence and defined WGM profiles.

FIG. 4 is a graphical representation showing experimental spectrashowing the observed shift in the WGM profile of microspheroidalparticle when a monolayer of a biopolymer is adsorbed to themicrospheroidal particles. QD=Quantum dot control uncoatedmicrospheroidal particle; PSS=microspheroidal particle with adsorbedmonolayer of polystyrene sulfonate (PSS); PVP=microspheroidal particlewith adsorbed monolayer of polyvinyl pyrrolidone (PVP).

FIG. 5 is a graphical representation of WGM profiles for: (a) DNA wasconjugated as 48-mer to Q-Sand particle; (b) Q-Sand beads hybridized toa-Transprobe DNA, the reverse complement of Transprobe; and (c) Q-Sandbeads hybridized to a PCR product.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides methods, inter alia, for detecting amolecule referred to herein as an analyte capable of entering into abinding interaction with another molecule referred to as its bindingpartner immobilized to the surface of a microspheroidal particle labeledwith an optically detectable label which confers on the particle adefined WGM profile. Reference to “optically detectable” includesreference to detection by spectrometric means. An interaction betweenthe analyte and the binding partner induces a change by the WGM profile.The methods described herein may be used for detecting analytes insamples and interactions between analytes. Importantly, neither theanalyte nor its binding partner require a label. The interaction inducesa change in the WGM profile of the microspheroidal particle. The methodsof the present invention are predicated, in part, on the phenomenon thatfluorescence emitters embedded within or onto a microspheroidal particleestablish defined WGM profiles. The microspheroidal particles may beexcited by a wide range of light sources. This facilitates measurementin many different configurations and facilitates multiplexing.

WGMs allow only certain wavelengths of light to be emitted from aparticle. The result of this phenomenon is that the usual broad emission(10-100 nm wide) bands from a fluorophore becomes constrained andappears as a series of sharp peaks corresponding effectively to standingmode patterns of light within the particle.

The WGM profile is extremely sensitive to interactions or associationswith the surface of a the microspheroidal particle. Hence, even rarebinding events can be detected due to the change in WGM profile.

It is to be understood that unless otherwise indicated, the subjectinvention is not limited to specific microspheroidal particleformulations, manufacturing methods, diagnostic or assay protocols, orthe like as such may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting. Reference to “ananalyte” and a “binding partner” is not to be inferred that anyknowledge of the structure of the analyte is known or required to beknown.

It must be noted that, as used in the subject specification, thesingular forms “a”, “an” and “the” include plural aspects unless thecontext already dictates otherwise. Thus, for example, reference to “apeak” or “a profile” includes a single peak or profile as well as two ormore peaks or profile; a “microspheroidal particle” includes a singleparticle as well as two or more particles; and so forth.

In one aspect, the present invention contemplates a method of detectingan analyte, said method comprising contacting at least one set ofmicrospheroidal particles with a sample putatively comprising saidanalyte, wherein each particle within a set of microspheroidal particlescomprises an optically detectable label and an immobilized putativebinding partner of said analyte wherein each particle set has a definedWhispering Gallery Mode (WGM) profile, wherein binding of said analyteto said immobilized binding partner results in a change in said WGMprofile of said at least one set of microspheroidal particles which isindicative of the presence of said analyte.

“Microspheroidal particles” contemplated by the present inventioninclude particles comprising any material, homogenous or otherwise whichcan produce one or more WGM profiles based on its optically detectablelabel. As will be evident to those of skill in the art, almost anymaterial, homogenous or otherwise may be used for the microspheroidalparticle. The microspheroidal particles contemplated herein may alsocomprise more than one substance, and as such may comprise shells,alloys or mixtures of organic and/or inorganic substances. It isadvantageous for quantification of the data generated by the methods ofthe present invention if the microspheroidal particle comprises asubstantially homogenous material with an isotropic refractive index andwhich is also non-absorbing (other than the optically detectable label,which is further described below).

Particularly useful materials which may be used in accordance with thepresent invention and which represent specific embodiments of thepresent invention include materials selected from the list consistingof: silica (for example: quartz or glass), latex, titania, tin dioxide,yttria, alumina, and other binary metal oxides (such as ZnO),perovskites and other piezoelectric metal oxides (such as BaTiO₃), ZnS,sucrose, agarose and other polymeric beads. In a particularly preferredembodiment, the “particle” and/or “microspheroidal particle” comprisessilica.

In addition, the particles contemplated by the present invention may beproduced in any convenient regular or irregular 3-dimensional shape.However, while many shapes of material can sustain WGM profile, it isgenerally practical to synthesize small spheres or spheroidal particles.Such spheres or spheroidal particles are also referred to herein as“microspheroidal particles” or “microspheres”. Accordingly, in preferredembodiments of the present invention, the “particles” and“microspheroidal particles” of the present invention are substantiallyspherical or spheroidal or comprise a “microsphere”.

Although the particles of the present invention may be referred to as“microspheroids” the actual size of the microspheroidal particle dependson a variety of factors and the particles may or may not actuallycomprise measurements in the micrometer range. In one preferredembodiment the microspheroidal particles of the present inventioncomprise a diameter (or equivalent measurement in a non-spheroidalparticle) of about 1 μm to about 100 μm, including 1 μm, 2 μm, 3 μm, 4μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, 21 μm, 22 μm, 23 μm, 24 μm, 25μm, 26 μm, 27 μm, 28 μm, 29 μm, 30 μm, 31 μm, 32 μm, 33 μm, 34 μm, 35μm, 36 μm, 37 μm, 38 μm, 39 μm, 40 μm, 41 μm, 42 μm, 43 μm, 44 μm, 45μm, 46 μm, 47 μm, 48 μm, 49 μm, 50 μm, 51 μm, 52 μm, 53 μm, 54 μm, 55μm, 56 μm, 57 μm, 58 μm, 59 μm, 60 μm, 61 μm, 62 μm, 63 μm, 64 μm, 65μm, 66 μm, 67 μm, 68 μm, 69 μm, 70 μm, 71 μm, 72 μm, 73 μm, 74 μm, 75μm, 76 μm, 77 μm, 78 μm, 79 μm, 80 μm, 81 μm, 82 μm, 83 μm, 84 μm, 85μm, 86 μm, 87 μm, 88 μm, 89 μm, 90 μm, 91 μm, 92 μm, 93 μm, 94 μm, 95μm, 96 μm, 97 μm, 98 μm, 99 μm and 100 μm. Reference to these sizes ofmircospheres include fractions of the whole numbers. For example,fractions between 1 and 2 μm include 1.05, 1.1, 1.15, 1.2, 1.25, 1.3,1.35, 1.4, 1.45, 1.5, 1.55, 1.6, 1.65, 1.7, 1.75, 1.8, 1.85, 1.9, 1.95or 2.0.

In a particular embodiment, the microspheroidal particles aremicrospheres.

A set of microspheroidal particles including a set of microspheresincludes a multiplicity (i.e. two or more) particles or microsphereshaving a common label or size or immobilized binding partner.

As used herein, the term “optically detectable label” refers to anymolecule, atom or ion which emits fluorescence, phosphorescence and/orincandescence. The optically detectable label may be chosen to emit atany wavelength at which WGM profile may be easily resolved. This dependson the ratio of the wavelength of the emission to the particle radius.Given that the sphere radius is arbitrary, the emission may be suitablychosen from the ultraviolet (wavelength range of about 350 nm to about 3nm), visible (wavelength range of about 350 nm to about 800 nm, nearinfrared (NIR) (wavelength range of about 800 nm to about 1500 nm)and/or infrared (IR) (wavelength range of about 1500 nm to about 101 m)ranges. However, due to the ease of detection, in one particularlypreferred embodiment, the optically detectable label is detectable inthe visible wavelength range.

In one particular embodiment, the optically detectable label may be anoptically detectable label which emits visible radiation in response toInfrared excitation. Such optically detectable labels are also referredto herein as “upconverters”.

Accordingly, another aspect of the present invention provides a methodof detecting an analyte, said method comprising contacting at least oneset of microspheroidal particles with a sample putatively comprisingsaid analyte, wherein each particle within a set of microspheroidalparticles comprises a label with emits visible radiation in response toinfrared excitation and an immobilized putative binding partner of saidanalyte wherein each particle set has a defined Whispering Gallery Mode(WGM) profile, wherein binding of said analyte to said immobilizedbinding partner results in a change in said WGM profile of said at leastone set of microspheroidal particles which is indicative of the presenceof said analyte.

The only constraint on the optically detectable label is that theemission in the chosen label should result in cavity mode emission.

In further embodiments of the subject invention, the opticallydetectable label comprises one or more labels selected from the listconsisting of a fluorophore, a semiconductor particle, phosphorparticle, a doped particle, or a nanocrystal and a quantum dot.Furthermore, the scattered light from small metal particles (surfaceplasmon emission) may be used as an optically detectable label.

Accordingly, a further aspect of the present invention is directed to amethod of detecting an analyte, said method comprising contacting atleast one set of microspheroidal particles with a sample putativelycomprising said analyte, wherein each particle within a set ofmicrospheroidal particles comprises an optically detectable labelselected from the list consisting of fluorophore, a semiconductorparticle, phosphor particle, a doped particle, or a nanocrystal and aquantum dot and an immobilized putative binding partner of said analytewherein each particle set has a defined Whispering Gallery Mode (WGM)profile, wherein binding of said analyte to said immobilized bindingpartner results in a change in said WGM profile of said at least one setof microspheroidal particles which is indicative of the presence of saidanalyte.

In another embodiment of the present invention, the optically detectablelabel is a fluorophore. As used herein, the term “fluorophore” refers toany molecule which exhibits the property of fluorescence. For thepurposes herein, the term “fluorescence” may be defined as the propertyof a molecule to absorb light of a particular wavelength and re-emitlight of a longer wavelength. The wavelength change relates to an energyloss that takes place in the process. The term “fluorophore” mayencompass a range of optically detectable labels such as chemicalfluorophores and dyes as well as quantum dots.

In a particular embodiment, the present invention provides a method ofdetecting an analyte, said method comprising contacting at least one setof microspheroidal particles with a sample putatively comprising saidanalyte, wherein each particle within a set of microspheroidal particlescomprises a fluorophore in the form of a quantum dot and an immobilizedputative binding partner of said analyte wherein each particle set has adefined Whispering Gallery Mode (WGM) profile, wherein binding of saidanalyte to said immobilized binding partner results in a change in saidWGM profile of said at least one set of microspheroidal particles whichis indicative of the presence of said analyte.

One particularly convenient optically detectable label which may be usedin accordance with the present invention is to embed fluorescentparticles on or in the microspheroidal particle. These opticallydetectable label particles may be so small that their properties andemission become size dependent. Such small optically detectable labelparticles are referred to in the art as semiconductor nanoparticles,quantum dots, quantum wires, quantum rods or nanocrystals orQ-particles. However, as used herein, the term “auantum dot” or “QD” isto be understood to encompass all such particles. Furthermore, opticallydetectable labels comprising QDs may comprise approximately spherical orspheroidal particles, or coated spherical or spheroidal particles.However, the term QD should not be considered in any way to be limitedto a spherical, spheroidal, circular, cylindrical or any othermorphology of a “dot”. For example, as used herein QDs may also compriseother morphologies including, inter alia, rod-like, ellipsoidal, orcoated rod-like or ellipsoidal particles.

QDs consist of a nanometer-scale crystalline core of semiconductormaterial; biologically active versions are typically surrounded by aprotective shell and external coat. For example, QDs may comprisesemiconductor crystallites which are about 2 nm to about 30 nm indiameter (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nm) and may containapproximately 50-500,000 atoms within the crystal, including luminescentcrystals comprising materials such as ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe,PbS, PbSe, PbTe, HgS, HgSe, HgTe, Si, ZnO.

QDs fluoresce with a broad absorption spectrum and a narrow emissionspectrum. Unlike some other fluorophores, which have distinct absorptionspectra, QDs absorb light over a wide spectral range, which allowsquantum dots to be excited with a range of light sources, such aslasers, arc lamps, or LEDs. Furthermore, a collection of different QDscan be used in multiplex applications using only a single excitationsource. However, the emission spectra for each dot is typically verynarrow, in the order of about 30 nm, the exact color depending on theparticle's diameter and composition. Furthermore, the narrow emissionspectrum of QDs permits spectral resolution of adjacent dots. Inaddition to the benefits above, QDs are also relatively photostable,even during intense excitation, and are brighter than fluorophores.

In light of the foregoing, it should also be understood that the presentinvention encompasses the use of different sized QDs in order tooptimise the wavelengths at which WGM profiles may be generated in amicrospheroidal particle.

Furthermore, the present invention contemplates QDs which are treatedwith procedures such as thermal treatment, surface modification,alloying, surface passivation or capping with surface coatings to enablethe QD to emit with high quantum yield and to improve the photostabilityfor long periods of time.

QDs are also commercially available from companies such as Quantum DotCorp. (QDC), which produces QDs such as the Qdot [Trade Mark] 605streptavidin conjugate, containing a cadmium-selenide core that emits at605 nm. Qdot conjugates that emit at 525, 565, 585, and 655 nm are alsoavailable. However, it should be understood that the present inventionis not limited in any way by the particular composition of the QD (orany other optically detectable label) and any QD (commercial orotherwise) may be compatible with the present invention.

There are also many fluorescent dyes which are available in the artwhich may be used as fluorophores in accordance with the presentinvention. An important property of a fluorescent dye or otherfluorophore, which determines it's potential for use is the excitationwavelength of the fluorophore; it must match the available wavelengthsof the light source. However, many different fluorescent dyes and otherfluorophores will be familiar to those of skill in the art, and thechoice of fluorescent marker in no way limits the subject invention.

Convenient “fluorophores” which may be used for the labeling of amicrospheroidal particle comprise any fluorescent marker which isexcitable using a light source selected from the group below:

-   -   (i) Argon ion lasers—comprise a blue, 488 nm line, which is        suitable for the excitation of many dyes and fluorochromes that        fluoresce in the green to red region. Tunable argon lasers are        also available that emit at a range of wavelengths (458 mn, 488        nm, 496 mn, 515 mn amongst others).    -   (ii) Diode lasers—have an emission wavelength of 635 nm. Other        diode lasers which are now available operate at 532 nm. This        wavelength excites propidium iodide (PI) optimally. Blue diode        lasers emitting light around 476 nm are also available. Such        diode lasers may be conveniently employed to excite WGMs within        the microspheroidal particles.    -   (iii) HeNe gas lasers—operate with the red 633 nm line. Such        lasers may be conveniently employed to excite WGMs within the        microspheroidal particles.    -   (iv) HeCd lasers—operate at 325 nm. Such lasers may be        conveniently employed to excite WGMs within the microspheroidal        particles.    -   (v) 100 W mercury arc lamp—the most efficient light source for        excitation of UV dyes like Hoechst and DAPI.    -   (vi) Xe arc lamps and quartz halogen lamps may likewise be used        as a means to excite WGMs and hence utilize the particles as        sensors.

In a particular embodiment of the present invention, the fluorescentmarkers are selected from: Alexa Fluor dyes; BoDipy dyes, includingBoDipy 630/650 and BoDipy 650/665; Cy dyes, particularly Cy3, Cy5 and Cy5.5; 6-FAM (Fluorescein); Fluorescein dT; Hexachlorofluorescein (HEX);6-carboxy-4′, 5′-dichloro-2′, 7′-dimethoxyfluorescein (JOE); Oregongreen dyes, including 488-X and 514; Rhodamine dyes, including RhodamineGreen, Rhodamine Red and ROX; Carboxytetramethylrhodamine (TAMRA);Tetrachlorofluorescein (TET); and Texas Red.

Two dyeing techniques are commonly used to fluorescently labelmicrospheroidal particles internal dyeing and external dyeing(surface-labeling). The two techniques produce particles with uniqueproperties, each beneficial for different applications. Internal dyeingproduces extremely stable particles with typically narrow fluorescenceemissions. These particles often display a greater resistance tophotobleaching. As the fluorophore is inside the beads, surface groupsare available for use in conjugating ligands (proteins, antibodies,nucleic acids, etc.) to the surface of the bead. For this reason,internally labeled beads are typically used in analyte-detection andimmunoassay applications. Surface-labeling involves conjugation of thefluorophore to the microspheroidal particle surface. Because thefluorophores are on the surface of the particle, they are able tointeract with their environment just as the fluorophores on a stainedcell. The result is a particle standard that exhibits the sameexcitation and emission properties as stained cell samples, under avariety of different conditions, such as the presence of contaminants orchanges in pH. The “environmentally responsive” nature ofsurface-labeled particles makes them ideally suited for mimickingbiological samples. Externally labeled particles are frequently used ascontrols and standards in a number of applications utilizingfluorescence detection. However, the present invention contemplates theassociation of a particle with a fluorescent label via any means.

The terms “phosphorescent particles”, “phosphor particles” and“phosphors” are used interchangeably herein. What constitutes aphosphorescent optically detectable label would be readily understood byone of skill in the art. However, by way of example, which in no waylimits the invention, suitable phosphors include small particles of ZnS,ZnS:Cu, Eu oxide and other phosphors used in display devices.

A optically detectable label comprising a “doped particle” may include aparticle which comprises occluded amounts of one or more rare earthions, such as Eu, Y, Yb, Sm and the like.

As used herein, the term “optically detectable label” should beunderstood to also encompass multiple optically detectable labels,mixtures of optically detectable labels, coated nanocrystals, alloys andother complex mixtures that would be evident to the skilled artisan. Theuse of all such optically detectable labels on microspheroidal particlesis to be considered as being within the scope of the methods andreagents described herein.

In accordance with the present invention, it has been shown that theemission of any particular label depends on the distribution of thelabel in the microspheroidal particle, the type of label and theconcentration of label. However, the methods of the present inventionare still practicable irrespective of whether the optically detectablelabel is at the surface of the microspheroidal particle, present as ashell within the microspheroidal particle, located at the core of themicrospheroidal particle or is present in more than one of the recitedlocations.

It should be noted that the methods of the present invention are notpredicated on quenching of the emission from the optically detectablelabel. The methods of the present invention, however, are predicated, inpart, on a modulation (i.e. a change) in the WGM profile of theoptically detectable label as a result of a interaction or associationof an analyte with a binding partner immobilized to the surface of amicrospheroidal particle.

WGMs, when dealing with electromagnetic radiation, are electromagneticresonances that can be established when incident light interacts with aparticle of higher refractive index than its surrounding medium. WGMsoccur at particular resonant wavelengths of light for a given particlesize, and the nature of the WGM may change with, inter alia, the size ofthe particle containing the WGM and the refractive indices of both theparticle and the surrounding medium. Furthermore, the size of theparticle can also effect the WGM established therein. WGMs areestablished when the incident light undergoes total internal reflectionat the particle surface.

Total internal reflection (TIR) may occur at the interface between twonon-absorbing media. When a beam of light propagating in the medium ofhigher refractive index meets an interface at a medium of lowerrefractive index at an angle of incidence above a critical angle, thelight is totally reflected at the interface and propagates back into thehigh refractive index medium. As will be evident to a person skilled inthe art, in a 3-dimensional medium the light may be reflected many timeswithin the particle of higher refractive index. In a WGM, the light isconcentrated near the circumference of the particle and can be assigneda mode number and a mode order. The mode number, n, provides the numberof wavelengths around the circumference of the particle, and the modeorder, l, provides the number of maxima in the radial dependence of theelectromagnetic field within the particle.

Fluorescence emitters embedded on or within a particle, as definedherein, display defined WGM profiles. These modes allow only certainwavelengths of light to be emitted from the particle. The result of thisphenomenon is that the usual relatively broad emission spectrum of anoptically detectable label (for example, fluorophores typically emit ina 10-100 nm wide band) becomes constrained and appears as a series ofsharp “peaks” corresponding effectively to standing mode patterns oflight within the particle. The series of peaks generated as a result ofthe establishment of a WGM in the microspheroidal particle of thepresent invention are referred to herein as “Whispering Gallery ModeProfiles” or “WGM Profiles”.

The WGM profile is extremely sensitive to both the position of theembedded optically detectable label and their concentration and spatialconfiguration with respect to each other. Furthermore, particle size andrefractive index are also important in determining the emissionwavelengths seen in a WGM profile.

It is proposed that the position and amplitude of one or more peaks in aWGM profile may be strongly influenced by interactions or associationsof the microspheroidal particle with molecules in a sample or externalenvironment.

In one example, association or binding of a molecule to amicrospheroidal particle alters the effective refractive index of themicrospheroidal particle altering the WGM profile generated by themicrospheroidal particle.

The term “refractive index” would be readily understood by a person ofskill in the art, Briefly, the refractive index of a medium is a valuecalculated from the ratio of the speed of light in a vacuum to that in asecond medium of greater density. In the case of a microspheroidalparticle, a change in “effective refractive index” may be a change inthe refractive index of the complete microspheroidal particle, or achange in effective refractive index may be a change in the refractiveindex of a region or part of the microspheroidal particle. For example achange in the effective refractive index of a microspheroidal particlemay comprise a change in the refractive index of the surface and/orperiphery of the microspheroidal particle.

In one particular embodiment, the present invention contemplates amethod for detecting the binding or association of a molecule to, orproximal with, the surface or sub-surface of the microspheroidalparticle wherein the binding or association of the molecule to animmobilized binding partner of the molecule effects a change in theeffective refractive index at the surface of the microspheroidalparticle.

A “sub-surface” includes pockets or pores surrounding the particle orwhich form a co-continuous interface between an internal environment ofthe particle and an external environment.

In yet another aspect, the present invention relates to the applicationof the method of the present invention for the detection of changes ofthe morphology, shape or size of a microspheroidal particle, wherein themicrospheroidal particle comprises a particle labeled with an opticallydetectable label and carries a molecule immobilized to the surface orsub-surface of the particle wherein said microspheroidal particleexhibits one or more WGM profiles and wherein one or more WGM profilesbased on the label of said microspheroidal particle detectably alterwith a change in the morphology, shape or size of the microspheroidalparticle when an analyte interacts with the immobilized molecule; themethod comprising:

-   -   (i) determining the initial WGM profile of the microspheroidal        particle;    -   (ii) subjecting the microspheroidal particle to a putative        change in the morphology, shape or size of the microspheroidal        particle following a potential interaction between an analyte        and the immobilized molecule;    -   (iii) determining one or more subsequent WGM profiles of the        microspheroidal particle;

wherein modulation in one or more subsequent WGM profile of themicrospheroidal particle, relative to the initial WGM profile, isindicative of a change of the morphology, shape or size of themicrospheroidal particle and the presence of an analyte associated withthe immobilized molecule.

As referred to herein the “morphology, shape or size” of amicrospheroidal particle refers to the spatial dimensions of themicrospheroidal particle. Accordingly, changes to the morphology, shapeor size of a microspheroidal particle include changes in any spatialdimension of the microspheroidal particle including, but not limited tochanges in height, width, depth, radius, diameter, circumference and thelike.

In yet another aspect, the present invention is directed to amicrospheroidal particle comprising a particle labeled with opticallydetectable label, wherein the microspheroidal particle exhibits a WGMprofile and wherein the WGM profile of the microspheroidal particledetectably modulates when an analyte interacts with a moleculeimmobilized to said microspheroidal particle.

As used herein, the molecule or binding partner immobilized to themicrospheroidal particle or the analyte in a sample refers to anychemical entity. Such chemical entities include, but are not limited tosmall chemical molecule; peptides, polypeptides and proteins or analogsthereof, nucleic acid molecules or analogs thereof, metal atoms or ionsor compounds comprising such atoms or ions. Of the nucleic acidmolecules, short interfering RNAs (single or double stranded) [siRNAs],interfering RNA complexes (RNAi) and DNA and RNA oligonucleotides areparticularly contemplated. Chemical compounds include entities producedin a combinatorial library or in a chemical library. In addition,natural product screening is contemplated from a biological source.Reference to a biological source includes an environmental sample,organism extract, plant or animal extract, serum, urine, exudate, semen,plasma, soil sample, river or sealed sample, extra-terrestrial sample,amongst other sources.

As used herein the phrase “bound to, or otherwise associated with”refers to any process by which a molecule may be associated with amicrospheroidal particle. Exemplary modes by which such associations maybe mediated include, but are not limited to: covalent binding, hydrogenbonding, van der Waals forces, ionic bonding, metallic bonding, polarbonding and dative (covalent) bonding and the like.

A molecule including a binding partner may also be attached to amicrospheroidal particle via an agent that promotes or increases theadsorption or binding of the molecule to the surface of themicrospheroidal particle, such an agent is referred to herein as a“linker”. For example, polynucleotides may be associated with amicrospheroidal particle via a linker which comprises a thiol, amine orcarboxyl group. Examples of suitable linkers include amino-terminatedsilanes such as amino-propyltrimethoxysilane oramino-propyltriethoxysilane. In addition to silanes, compounds such aspoly-L-lysine that non-covalently attach to surfaces such as glass andelectrostatically adsorb the phosphate groups of a polynucleotide arealso within the scope of the present invention. Therefore, othermolecules, including other silanes, which are suitable to promote thebinding or association of a polynucleotide, polypeptide or othercompound to the surface of a microspheroidal particle would be readilyidentified by the skilled artisan, and the present invention is notlimited by the choice of linker.

In one embodiment, the present invention contemplates a microspheroidalparticle comprising a particle labeled with an optically detectablelabel, wherein said microspheroidal particle exhibits a WGM profile andwherein the WGM profile of the microspheroidal particle detectablymodulates when a molecule binds or associates with the microspheroidalparticle, such as via a molecule comprising a nucleic acid moleculebound to, or otherwise associated with, the surface of themicrospheroidal particle.

The terms “nucleic acids”, “nucleotide” and “polynucleotide” includeRNA, cDNA, genomic DNA, synthetic forms and mixed polymers, both senseand antisense strands, and may be chemically or biochemically modifiedor may contain non-natural or derivatized nucleotide bases, as will bereadily appreciated by those skilled in the art. Such modificationsinclude, for example, labels, methylation, substitution of one or moreof the naturally occuring nucleotides with an analog (such as amorpholine ring), intemucleotide modifications such as unchargedlinkages (eg. methyl phosphonates, phosphotriesters, phosphoamidates,carbamates, etc.), charged linkages (eg. phosphorothioates,phosphorodithioates, etc.), pendent moieties (eg. polypeptides),intercalators (eg. acridine, psoralen, etc.), chelators, alkylators andmodified linkages (eg. α-anomeric nucleic acids etc.). Also included aresynthetic molecules that mimic polynucleotides in their ability to binda designated sequence via hydrogen bonding and other chemicalinteractions. Such molecules are known in the art and include, forexample, those in which peptide linkages substitute for phosphatelinkages in the backbone of the molecule.

Another embodiment of the present invention contemplates amicrospheroidal particle as hereinbefore described wherein themicrospheroidal particle further comprises DNA bound to, or otherwiseassociated with, the surface of the microspheroidal particle.

Still a further preferred embodiment of the present inventioncontemplates a microspheroidal particle as hereinbefore describedwherein the microspheroidal particle further comprises RNA or a complexof RNA (e.g. RNA—Rnase complex) bound to, or otherwise associated with,the surface of the microspheroidal particle.

In an alternative embodiment, the present invention contemplates amicrospheroidal particle comprising a particle labeled with opticallydetectable label, wherein the microspheroidal particle exhibits a WGMprofile and wherein the WGM profile of the microspheroidal particledetectably modulates when a peptide, polypeptide or protein or analogthereof binds to, or otherwise associated with, the surface of themicrospheroidal particle.

In one embodiment, the peptide, polypeptide or protein is an enzyme.

In another embodiment, the peptide, polypeptide or protein is anantibody.

The term “antibody” refers to a protein of the immunoglobulin familythat is capable of combining, interacting or otherwise associating withan antigen. An antibody is, therefore, an antigen-binding molecule. Theterm “antigen” is used herein in its broadest sense to refer to asubstance that is capable of reacting with or binding to theantigen-binding site of an antibody. With reference to the presentinvention, an antigen also includes the idiotype of an antibody.

The term “immunoglobulin” is used herein to refer to a proteinconsisting of one or more polypeptides substantially encoded byimmunoglobulin genes. The recognized immunoglobulin molecules includethe κ, λ, α, γ (IgG₁, IgG₂, IgG₃, IgG₄), δ, ε and μ constant regions,light chains (κ and 1), as well as the myriad immunoglobulin variableregions. One form of immunoglobulin constitutes the basic structuralunit of an antibody. This form is a tetramer and consists of twoidentical pairs of immunoglobulin chains, each pair having one light andone heavy chain. In each pair, the light and heavy chain variableregions are together responsible for binding to an antigen, and theconstant regions are responsible for the antibody effector functions. Inaddition to antibodies, immunoglobulins may exist in a variety of otherforms including, for example, Fv, Fab, Fab′ and (Fab′)₂ and chimericantibodies and all of these variants are encompassed by the term“antibody” as used herein. In addition, immunoglobulins from otheranimals (eg. birds, mammals, fish, amphibians, and reptiles) havesimilar function, but different nomenclature and these are considered“antibodies ” as well.

The present invention also contemplates microspheroidal particlescomprising anti-idiotypic antibodies bound to, or otherwise associatedtherewith. As used herein, the term “anti-idiotypic antibody” refers toan antibody which recognizes and binds to antigenic determinants withinthe variable region, for example the V_(H) and/or V_(L) domains, of atarget antibody. These antigenic determinants within the variable regionof an antibody are referred to as the “idiotype” of an antibody.Accordingly, an antibody specific for these regions is referred to as an“anti-idiotypic antibody”. “Analogs” of peptides, polypeptides and/orproteins contemplated herein include but are not limited to peptides,polypeptides or proteins comprising modification to side chains,synthetic peptides that incorporate unnatural amino acids and/or theirderivatives during synthesis and the use of crosslinkers and othermethods which impose conformational constraints on the subject peptide,polypeptide or protein.

Examples of side chain modifications include modifications of aminogroups such as by reductive alkylation by reaction with an aldehydefollowed by reduction with NaBH₄; amidination with methylacetimidate;acylation with acetic anhydride; carbamoylation of amino groups withcyanate; trinitrobenzylation of amino groups with 2,4,6-trinitrobenzenesulphonic acid (TNBS); acylation of amino groups with succinic anhydrideand tetrahydrophthalic anhydride; and pyridoxylation of lysine withpyridoxal-5 -phosphate followed by reduction with NaBH₄.

The guanidine group of arginine residues may be modified by theformation of heterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation viaO-acylisourea formation followed by subsequent derivitization, forexample, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylationwith iodoacetic acid or iodoacetamide; performic acid oxidation tocysteic acid; formation of a mixed disulphide with other thiolcompounds; reaction with maleimide, maleic anhydride or othersubstituted maleimide; formation of mercurial derivatives using4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid,phenylmercury chloride, 2-chloromercuri-4-nitrophenol and othermercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation withN-bromosuccinimide or alkylation of the indole ring with2-hydroxy-5-nitrobenzyl bromide or sulphonyl halides. Tyrosine residueson the other hand, may be altered by nitration with tetranitromethane toform a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may beaccomplished by alkylation with iodoacetic acid derivatives orN-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives duringpeptide synthesis include, but are not limited to, use of norleucine,4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid,6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine,omithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienylalanine and/or D-isomers of amino acids. A list of unnatural amino acid,contemplated herein is shown in Table 2.

TABLE 2 Codes for non-conventional amino acids Non-conventionalNon-conventional amino acid Code amino acid Code α-aminobutyric acid AbuL-N-methylalanine Nmala α-amino-α-methylbutyrate MgabuL-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagineNmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid AibL-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmglncarboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine ChexaL-Nmethylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucineNmile D-alanine Dal L-N-methylleucine Nmleu D-arginine DargL-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmethionine NmmetD-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine DglnL-N-methylnorvaline Nmnva D-glutamic acid Dglu L-N-methylornithine NmornD-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine DileL-N-methylproline Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysineDlys L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophanNmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine DpheL-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine NmetgD-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine DthrL-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyrα-methyl-aminoisobutyrate Maib D-valine Dval α-methyl-γ-aminobutyrateMgabu D-α-methylalanine Dmala α-methylcyclohexylalanine MchexaD-α-methylarginine Dmarg α-methylcylcopentylalanine McpenD-α-methylasparagine Dmasn α-methyl-α-napthylalanine ManapD-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteineDmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine DmglnN-(2-aminoethyl)glycine Naeg D-α-methylhistidine DmhisN-(3-aminopropyl)glycine Norn D-α-methylisoleucine DmileN-amino-α-methylbutyrate Nmaabu D-α-methylleucine Dmleu α-napthylalanineAnap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionineDmmet N-(2-carbamylethyl)glycine Ngln D-α-methylornithine DmornN-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine DmpheN-(2-carboxyethyl)glycine Nglu D-α-methylproline DmproN-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycineNcbut D-α-methylthreonine Dmthr N-cycloheptylglycine NchepD-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-α-methyltyrosineDmty N-cyclodecylglycine Ncdec D-α-methylvaline DmvalN-cylcododecylglycine Ncdod D-N-methylalanine Dnmala N-cyclooctylglycineNcoct D-N-methylarginine Dnmarg N-cyclopropylglycine NcproD-N-methylasparagine Dnmasn N-cycloundecylglycine NcundD-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine NbhmD-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine NbheD-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine NargD-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine NthrD-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine NserD-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine NhisD-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvalD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomophenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetL-α-methylleucine Mleu L-α-methyllysine Mlys L-α-methylmethionine MmetL-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithineMorn L-α-methylphenylalanine Mphe L-α-methylproline MproL-α-methylserine Mser L-α-methylthreonine Mthr L-α-methyltryptophan MtrpL-α-methyltyrosine Mtyr L-α-methylvaline MvalL-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl) NnbhmN-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycinecarbamylmethyl)glycine 1-carboxy-1-(2,2-diphenyl- Nmbcethylamino)cyclopropane

Crosslinkers can be used, for example, to stabilize 3D conformations,using homo-bifunctional crosslinkers such as the bifunctional imidoesters having (CH₂)_(n) spacer groups with n=1 to n=6, glutaraldehyde,N-hydroxysuccinimide esters and hetero-bifunctional reagents whichusually contain an amino-reactive moiety such as N-hydroxysuccinimideand another group specific-reactive moiety such as maleimido or dithiomoiety (SH) or carbodiimide (COOH). In addition, peptides can beconformationally constrained by, for example, incorporation of C_(α) andN_(α)-methylamino acids, introduction of double bonds between C_(α) andC_(β) atoms of amino acids and the formation of cyclic peptides oranalogs by introducing covalent bonds such as forming an amide bondbetween the N and C termini, between two side chains or between a sidechain and the N or C terminus.

In yet another alternate preferred embodiment, the present inventioncontemplates a microspheroidal particle comprising a particle labeledwith optically detectable label, wherein the microspheroidal particleexhibits a WGM profile and wherein the WGM profile of themicrospheroidal particle detectably modulates when a carbohydratemolecule or analog thereof binds to, or otherwise associates with, thesurface of the microspheroidal particle.

In one particularly preferred embodiment, the carbohydrate molecule is aGlycosaminoglycan (GAG) molecule or GAG-like molecule.

GAGs are ubiquitous and play pivotal roles in many of the inflammatoryprocesses within the human body. These large molecular weightpolysaccharides contribute to such processes as cancer metastasis,arthritis, transplant rejection and asthma and thus a greaterunderstanding of these processes may lead to improved drugs for theeventual treatment of such conditions. Currently, one of the best knownGAGs is the heparin family of sulfated polysaccharides and theanti-coagulant activity of these molecules is well understood.

Heparin-Like GAGs (HLGAGs) are a heterogenous group of molecules(Conrad, Heparin binding proteins. Academic Press, San Diego, 1998;Lander and Selleck, J Cell Biol 148(2):227-232, 2000). Heparin andheparan sulfate, like all HLGAGs, are long linear polysaccharides(Sasisekharan and Venkataraman, Curr Opin Chem Biol 4(6):626-631, 2000;Casu, Ann NY Acad Sci 556:1-17, 1989; Casu, Adv Carbohydr Chem Biochem43:51-134, 1985). They are synthesized as non-sulfated chains ofrepeating disaccharide units comprising glucuronic acid (GlcA) andglucosamine (GlcN) which, in the golgi, are modified at various sitesalong their length. Heparin is more extensively modified than heparansulfate and most of the GlcN units are modified by a sulfate group tobecome N-sulfated GlcN and most GlcA units are converted to iduronicacid (IdoA) through the action of epimerase. HLGAGs are heterogenoussince modifications to the sulfate chains are often incomplete. Theresult is extensive regions of intermediate modification.

Thus, for example, heparan sulfate chains consist of highly sulfated,structurally flexible domains rich in 2-O-sulfated IdoA alternating withregions of low sulfation consisting predominantly of N-acetyl GlcN andGlcA, which are a rigid structure. The sulfation patterns of HLGAGs arecomplex especially with respect to the positioning of 6-O-sulfates.Consequently, not all HLGAG molecules are identical. It is the sulfationpattern which largely determines the protein binding characteristics ofa particular HLGAG.

Some proteins bind only to particular structural motifs within a HLGAGchain and conversely some GAGs bind only to particular sites or regionson a protein. Anti-thrombin III, for example, binds to a uniquepentasaccharide sequence displaying a particular arrangement of sulfategroups and the heparin pentasaccharide binds to a specific site on theanti-thrombin III protein (Whisstock et al. J Mol Biol 301:1287-1305,2000). Basic fibroblast-derived growth factor (FGF-2) and hepatocytegrowth factor (HGF) both bind heparin, but the heparin structures thatare essential for binding are quite different for each and are differentfrom that required by anti-thrombin III (Maccarana et al. J Biol Chem268(32):23898-23905, 1993; Lyon et al. J Biol Chem 269:11216-11223,1994). Moreover, heparin has been shown to bind to a particular regionon FGF-2 (Faham et al. Science 271:1116-1120, 1996).

FGF-2 recognizes a motif containing a single IdoA 2-O-sulfate in adefined position, whereas for HGF, the positioning of the GlcN6-O-sulfate groups are critical. Some heparin molecules within apreparation will carry both the anti-thrombin III bindingpentasaccharide and the FGF-2 binding motif, whereas others will carrythe HGF binding motif and the FGF-2 motif and not the anti-thrombin IIIbinding pentasaccharide. Indeed, on average only one third of themolecules within a preparation of heparin carry the anti-thrombin IIIbinding pentasaccharide (Conrad, 1998, supra).

GAG-like molecules may also be derived from non-mammalian sources. Forexample, the capsular polysaccharide from E. coli K5 is composed of analternate α-N-acetyl glucosamine (α-GlcNAc) and β-glucuronic acid(β-GlcA) units and contains no sulfate or other charged groups. Theheparin backbone consists of the following motif α-GlcNAc, β-GlcA,α-GlcNAc, β-iduronic acid (β-IdoA) with varying degrees of sulfation.The only difference between GlcA and IdoA is the configuration of thecarboxylic acid group at C-5, thus the heparin backbone and the E. coliK5 backbone are extremely similar in structure.

In yet another aspect, the present invention provides a method ofdetecting a molecule capable of entering into a binding interactionbetween a molecule immobilized to, or otherwise associated with, amicrospheroidal particle comprising an optically detectable label, ashereinbefore described, and a putative analyte binding partner of saidmolecule in a sample, said method comprising:

-   -   (i) determining a initial WGM profile of the microspheroidal        particle comprising the molecule immobilized to, or otherwise        associated therewith; and    -   (ii) contacting the microspheroidal particle comprising said        immobilized microspheroidal molecule or otherwise associated        therewith, with a sample comprising a putative analyte binding        partner of said molecule;

wherein a detectable change in the WGM profile of the microspheroidalparticle, relative the initial WGM profile, is indicative of binding orinteraction between the molecule and the analyte binding partner of themolecule.

As used herein “a detectable change in the WGM profile” may comprise anydifference that may be observed between any two WGM profiles.

In one preferred embodiment, the detectable change may comprise ared-shift or blue-shift of one or more peaks in one WGM profile relativeto another WGM profile.

As used herein the term “red-shift” refers to the shifting of the pointof maximum amplitude of one or more peaks in a WGM profile to a longerwavelength. Despite the name “red”, a red-shift may occur in any part ofthe electromagnetic spectrum and is not limited to visible light. Asused herein, the term “red-shift” includes any shifting of a peak to alonger wavelength. Conversely, the term “blue-shift” refers to anymovement of a peak to a shorter wavelength.

In a particularly preferred embodiment, the red-shift or blue-shift of apeak in a WGM profile typically comprises a change in the wavelength ofthe maximum amplitude of the peak of approximately 1 to 100 nm.Reference to 1 to 100 nm includes wavelengths of 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99 and 100 nm. In a particular embodiment, the red-shift or blue-shiftof a peak in a WGM profile comprises a change in the wavelength of themaximum amplitude of the peak of approximately 1 to 20 mn, whichincludes wavelengths of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19 and 20 nm. Although specifically exemplified withrespect to wavelength shifts in visible light peaks, the invention alsocontemplates equivalent and/or proportionate red-shifts and blue-shiftsin other regions of the electromagnetic spectrum.

In a further embodiment of the present invention, the change in one ormore WGM profiles of a microspheroidal particle, caused by aninteraction of an immobilized molecule on the microspheroidal particlewith an analyte may comprise the appearance of one or more peaks in oneor more of the WGM profiles or the disappearance of one or more peaks inone or more of the WGM profiles.

A WGM profile of a microspheroidal particle as described herein may beascertained using any convenient method that would be evident to one ofskill in the art. Essentially any detection method which can detect oneor more wavelengths of electromagnetic radiation may be used to detect aWGM profile. Preferably, the detection means is sufficiently sensitivesuch that it can differentiate peaks in the WGM profile, morepreferably, the detection means can differentiate between two peakswhich are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and/or 100 nm apart.Particularly convenient means which may be used to determine the WGMprofile of a microspheroidal particle include a flow-cytometer, arrayreader and a confocal microscope.

As used herein the term “confocal microscope” includes within its scope,laser scanning confocal microscopes (LSCM) and multiphoton confocalmicroscopes.

The present invention also provides a microspheroidal particleimmobilized to a solid support.

Accordingly, in another aspect, the present invention provides amicrospheroidal particle comprising a particle labeled with opticallydetectable label, wherein the microspheroidal particle exhibits a WGMprofile and wherein the WGM profile of the microspheroidal particledetectably modulates with said microspheroidal particle is immobilizedonto a solid support and/or an analyte interacts with a binding partnercaptured on the immobilized particle.

As used herein the term “solid support” refers to any solid matrix ontowhich a microspheroidal particle may be immobilized. In preferredembodiments of the invention, the solid support comprises a solidsupport which allows the WGM profile of the microspheroidal particleimmobilized thereto to be detected. Accordingly, preferable solidsupports are those which may be used to immobilize a microspheroidalparticle for analysis using a confocal microscope. In a particularlypreferred embodiment, the solid support is a microscope slide.

In yet another aspect, the present invention contemplates a microscopeslide comprising one or more indentations therein, wherein eachindentation is adapted to immobilize a microspheroidal particle asdescribed herein. Preferably, a microspheroidal particle is immobilizedonto said slide, by said particle settling into, or embedding into anindentation in said slide.

In yet another aspect, the present invention provides a method ofdetecting an analyte putatively in a sample, said method comprising:

-   -   (i) determining an initial WGM profile of a microspheroidal        particle comprising a particle labeled with optically detectable        label and a molecule immoblized to or associated therewith,        wherein the microspheroidal particle exhibits the WGM profile        and wherein the WGM profile of the microspheroidal particle        detectably modulates when said microspheroidal binds or        otherwise interacts with the analyte putatively in said sample;    -   (ii) applying said sample to said microspheroidal particle for a        time and under conditions to allow interaction of the analyte        with the molecule immobilized, or otherwise associated to the        microspheroidal particle;    -   (iii) subsequently determining the WGM profile of the        microspheroidal particle;

wherein a detectable change in a the WGM profile relative to an initialWGM profile is indicative of the presence of said analyte in saidsample, and an absence of a detectable change in a WGM profile relativeto an initial WGM profile is indicative of the absence of said analytein said sample.

In one embodiment the molecule bound to, or otherwise associated with,the surface of said microspheroidal particle and/or said analytecomprises a nucleic acid molecule. Even more preferably, the nucleicacid molecule is DNA or RNA.

In another preferred embodiment of this aspect of the present invention,the molecules bound to, or otherwise associated with, the surface of themicrospheroidal particle and/or said analyte comprises a peptide,polypeptide or protein or analog thereof as defined herein.

In yet another preferred embodiment of this aspect of the presentinvention, the molecule bound to, or otherwise associated with, thesurface of the microspheroidal particle and/or said analyte comprises acarbohydrate molecule. In an even more preferred embodiment, thecarbohydrate molecule is a GAG molecule.

In yet another preferred embodiment of this aspect of the subjectinvention, the molecule bound to, or otherwise associated with, thesurface of the microspheroidal particle, and/or said analyte comprises amolecule which binds to, or otherwise interacts with a nucleic acid,peptide, polypeptide, protein and/or carbohydrate molecule.

In yet another aspect of the invention, a method of identifying bindingpartners of a molecule of interest is provided, said method comprising:

-   -   (i) determining a initial WGM profile of a microspheroidal        particle comprising a particle labeled with optically detectable        label and a molecule immobilized to or associated to therewith;    -   (ii) applying one or more samples comprising potential binding        partners of said molecule of interest to the microspheroidal        particle for a time and under conditions to allow interaction of        the molecule of interest with the potential binding partner(s);    -   (iii) subsequently determining the WGM profile of the        microspheroidal particle;

wherein modulation of said WGM profile relative to an initial WGMprofile indicative of the presence of one or more binding partner(s) insaid sample, and an absence of a detectable change in the WGM profilerelative to the initial WGM profile is indicative of the absence of oneor more binding partner(s) in said sample.

In one preferred embodiment the molecule bound to, or otherwiseassociated with, the surface of the microspheroidal particle and/or saidanalyte comprises a nucleic acid molecule. Even more preferably, thenucleic acid molecule is DNA or RNA.

In another preferred embodiment of this aspect of the invention, themolecules bound to, or otherwise associated with, the surface of themicrospheroidal particle and/or said analyte comprises a peptide,polypeptide or protein or analog thereof as defined herein.

In yet another preferred embodiment of this aspect of the invention, themolecule bound to, or otherwise associated with, the surface of themicrospheroidal particle and/or said analyte comprises a carbohydratemolecule. In an even more preferred embodiment, the carbohydratemolecule is a GAG molecule.

In yet another preferred embodiment of this aspect of the instantinvention, the molecule bound to, or otherwise associated with, thesurface of the microspheroidal particle, and/or said analyte comprises amolecule which binds to, or otherwise interacts with a nucleic acid,peptide, polypeptide, protein and/or carbohydrate molecule.

The microspheroidal particles of the present invention can be used forthe following applications: environmental monitoring, such as waterquality (Legionella, Giardia, Cryptosporidium, at the source) and pointof care diagnostics, airborne pathogens and toxins, protein:proteininteraction screening, protein:carbohydrate interaction screening anddrug screening, and screening of libraries of small molecules.

The present invention enables multiplexing due to the ability to havetwo or more sets of microspheroidal particles. The particles in any oneset may have a common optically detectable label and/or a common sizeand/or a common immobilized binding particle. Hence, multiple analytesmay be detected using a multiplicity of sets of microspheroidalparticles.

The present invention further provides analytes detected by the methodof the present invention.

The present invention also further provides kits comprising labeled orpre-labeled microspheroidal particles. The particles may be used in thegeneration of an assay for detecting changes in an environmentsurrounding a particle.

The present invention is further described by the following non-limitingexamples:

EXAMPLE 1 Production and Labeling of Quantum Dot Labeled MicrospheroidalParticles

Typically, microspheroidal particles may be prepared by overlayingcommercial beads of any desired composition or size with quantum dots.For more robust materials, they should be overcoated with another silicalayer to minimize interactions with the medium or adsorbates.

Without limiting the many possibilities, described herein is a typicalprocess used to prepare microspheroidal particles using glass/silicabeads.

0.1 g of five micron diameter commercial silica beads were placed in 20ml 2-propanol and 20 microlitres of either mercaptopropylsilane (MPS) oraminopropylsilane (APS) was added. The solution was refluxed at 80° C.to allow chemisorption of the MPS or APS, which led to the creation ofmercaptan or amino groups on the bead surface. Excess MPS or APS wasremoved by centrifugation.

Instead of a silane functionalized bead, the beads can also be activatedby adsorbing a cationic polymer. For example without restriction and toillustrate the method, 0.1 g of silica beads were mixed with 20 mL of anaqueous PEI (poly(ethylene imine) solution (1 g/L, 0.5M NaCl). Afterreacting for one hour, the solution was centrifuged to remove the excesspolymer and resuspended in pure water. This procedure was repeated 3times.

QDs were adsorbed onto the APS or PEI coated beads by adding asuspension of QDs in chloroform to a suspension of the beads in dry2-propanol. The amount added was such as to coat the beads with about 10monolayers. The beads and QDs were equilibrated for 10 minutes in arotation tumbler. Then, the coated beads were separated bycentrifugation.

The QD labeled beads can be further stabilized by coating with moresilica. This was done in two ways:

First, 5 uL of APS in alkaline water or chloroform (1 mL) was added tothe QD coated silica microspheroidal particles and allowed to react for1 hour with agitation using a rotation tumbler at 0.2 Hz. After removingthe excess APS by repeated centrifugation/resuspension cycles inalkaline water or chloroform, 5 mL of “active silica” (5 uL of 2 w/w %sodium silicate in 5 mL of water at pH 8.7) was added to the particlesand allowed to react overnight. This resulted in about 3-5 nm of silicabeing deposited onto the particles. Excess nucleated free silica wasseparated via centrifugation and the particles redispersed in2-propanol:water (4:1). Finally the silica layer was increased usingTEOS and ammonia as required to obtain the desired thickness.

The second procedure involved direct silica growth in ethanol without apreliminary deposition step in water. This consists of adsorption of PVPand then growth of silica. Enough PVP (MW 30,000) was added to provide60PVP molecules per nm² of surface, by dissolving it inchloroform:2-propanol (9:1). This was immediately added to themicrospheres, the mixture was sonicated and allowed to react overnightunder stirring.

The microspheres were centrifuged (5 sec at 3800 rpm for 5 micronsilica) and re-dispersed in 2-propanol (first time). NH₃ (4.2%) followedby TEOS (10vol % in 2-propanol) was added with stirring and left todeposit over 12 hours.

A schematic showing the procedure for producing QD labeledmicrospheroidal particles is shown in FIG. 1. This generic protocolallows the preparation of photostable, silica microspheres of anypractical size from 1 micron up to 100 microns, labeled with one or moredifferent nanoparticles with different emission properties.

Examples of QD labeled microspheroidal particles as viewed under aconfocal microscope are shown in FIG. 2.

EXAMPLE 2 DNA Detection

Q-Sand beads were conjugated to DNA, forming a complex with a Q-Sandbead and immobilized 48-mer DNA. The fifth base position of the 48-merwas a thymidine base with an incorporated amine. This amine was used tolabel the DNA with a BODIPY·630/650 NHS ester using a standardcondensation reaction. The WGM profile for the Q-Sand bead conjugated tothe 48-mer DNA is shown in FIG. 5, panel a.

Panel b of FIG. 5 shows the WGM profile of the Q-Sand bead of panel aconjugated to a α-Transprobe DNA which hybridizes to bases 6-24 of theDNA conjugated to the Q-Sand bead. The resulting WGM profile, as seen inpanel b of FIG. 5, was a shift of approximately 2.4 nm for all peaks. Inaddition, the Q-factor (the measure of the quality of themicroresonator) reduction was approximately 5×.

Finally, the Q-Sand beads shown in FIG. 5, panel a, were hybridized to aPCR product containing a complementary region to DNA bases 25-48 of theDNA 48-mer. The PCR product was generated from HeLa cell DNA andprepared for hybridization by Exol and Lambda Exo digestion. Theresultant WGM profile was shifted 2.4 nm for all peaks. The Q-factor wasapproximately 10× drop from Q-Sand only control.

EXAMPLE 3 Surface Functionalization of Silica Microspheroidal Particles

Surface functionalization of silica microspheroidal particles can bedone in the same way used for the initial silica microspheroidalparticles by refluxing the QD-silica microspheroidal particles in2-propanol containing MPS or APS or other silane to activate the surfaceand add functional groups which react with the target bioadsorbate.

Preparation of Conjugates

5 micron microspheroidal particles labeled with orange QDs emitting at560 nm and overcoated with 10 nm silica were treated with MPS,centrifuged and washed. The microspheres were allowed to react in waterwith the conjugate, such as a nucleic acid, polypeptide, antibody,carbohydrate or the like for about 1 hour. They were then centrifugedand washed to yield Bioactive QD Microspheres (BQDM).

Binding Assays:

Several BQDM are placed on a microscope slide under a confocalmicroscope. A drop of reference solution is placed on the microspheresand the spectra collected using 488 nm laser excitation. Then amicrolitre of a solution containing an agent which putatively interactswith the conjugate on the BQDM is added to the drop and allowed to reactover half an hour. The spectrum is collected and any change in the WGMprofile, such as a red-shift or blue-shift of one or more peaks, isdetected, wherein an observed change is indicative of interactionbetween the conjugate on the microspheroidal particle and theexogenously added agent.

EXAMPLE 4 Differentiation of Microspheroidal Particles Using WGM ProfileDifferentiation by Size

Microspheroidal particles of different sizes were produced using themethods described herein. WGM profiles of each of the different particlesizes were determined using a confocal microscope.

As shown in FIG. 3, the WGM profile exhibited by each of the differentsized particles changes with the radius of the microspheroidal particle.

Differentiation by Compound Surface Compound

QD labeled microspheroidal particles comprising different moleculesbound to their surface were produced using the methods described herein.WGM profiles of each of the different microspheroidal particles weredetermined using a confocal microscope.

As shown in FIG. 4, QD refers to a QD labeled microspheroidal particlewith no compound bound to the surface, PSS refers to a microspheroidalparticle comprising bound polystyrene sulfonate (PSS), while PVP refersto microspheroidal particle with an adsorbed monolayer of polyvinylpyrrolidone (PVP).

As is evident from the data, the QD labeled microspheroidal particlesare sensitive to the binding of a compound to their surface.Furthermore, the WGM profile shift observed also appears sensitive tothe nature of the compound binding to the surface.

EXAMPLE 5 The Detection of a Putative Binding Partner in a Sample

Silica microspheres with functional surface sulfhydryl groups areconjugated, via a 5′ acrydite molecule, to single strandedoligonucletides, which have previously been labeled with a fluorescentdye.

The microspheres with attached, labeled oligonucleotide, are thenhybridised to complimentary or unrelated oligonucleotides or PCRproducts.

The hybridised and unhybridised microspheres are extensively washed inMilli Q water and spotted out on a microscope cover slip and allowed toair dry.

The dried microspheres are then examined on a confocal microscope withlaser and wavelength filters, appropriate for the fluorescent dyes beingused. The whispering gallery modes generated are recorded and comparedto determine the wavelength shift in the whispering gallery mode patternbetween, microspheres with additional DNA hybridised onto themicrosphere surface and microspheres with labeled oligonucleotideattached only.

The resulting shift in the wavelength pattern of the whispering gallerymodes can be potentially used to determine the presence or absence ofcomplementary DNA sequences in test samples.

EXAMPLE 6 Screening of Test Compounds

Microspheroidal particles are produced using the protocols describedherein. Different beads are identifable by WGM profile. Even thoughthere is some variability between beads of a particular type, the WGMprofile for a given Target with a given fluorescent emitter on a bead ofa given size will be almost equivalent. At least 50 different Q-Sandbeads can be tested simultaneously.

Microspheroidal particles are arrayed into reaction carriers. Thesecarriers are gridded glass slides with regions for different compounds.The grids are scanned and WGMs are calculated and saved. This reading isthe pre-compound control WGM.

Each reaction carrier is tested with one or more of test molecules.

Non-specific binding is alleviated by washing step(s).

After washing the reaction carrier is re-scanned. Positives areidentified by WGM shifts compared to pre-test molecule control WGMs.

EXAMPLE 7 Microsphere Synthesis Protocol Materials:

(3-aminopropyl)trimethoxysilane (APS 99%),(3-mercaptopropyl)trimethoxysilane (MPS, 95%), tetraethyl orthosilicate(TEOS, 98%), polyvinylpyrrolidone (PVP, MW 40,000), Ammonium Hydroxide(29.1% wt % NH3 water) (Sigma-Aldrich). 5 μm silica particles (BangsLaboratories, Inc.) Chloroform (CH3C13) and 2-propanol (AnalaR, Merck,Kilsyth, Victoria).

Instruments:

Olympus Fluorescence Microscope (Olympus, Bonn, Germany), MotorizedRotating Wheel, Shake and Stack (Thermohybaid, Glochester UK),Ultrasonic Cleaner (Cole-Parmer Instrument Company, Illinois, USA).

General Important Notes:

Sterilize all glass-vials and magnetic stirrers with ethanol and allowto completely Dry.

Use filtered tips for all pipetting.

All glass vials must be flat-bottomed and screw-cap between 1.5-5 mlvolume.

Para-film all glass screw cap reaction vials when reaction requiresstirring-overnight.

Surface functionalization of silica beads was prepared as prescribed byBrendan Toohey.

The ultrasonication during the wash steps and the dissolving of PVP isnot essential

Surface Functionalization Silica Beads

-   -   1. Thoroughly clean a round bottom flask x1 with acetone and x2        with 2-propanol.    -   2. Add 20 ml of 2-propanol to the cleaned round bottom flask and        set up in a heating mantle, use plastic lid clips to keep any        lids fastened and begin to push water through the shlenk line.    -   3. The 2-propanol is required to be heated to 80° C. under        constant stirring for reaction to take place optimally, thus        with a thermometer constantly monitor the temperature of the        2-propanol until it reaches temperature, make sure thermometer        is wiped with ethanol before each temperature check.    -   4. Once temperature is reached add 20 μl of APS and 0.1 g of        untreated Bang's Beads which equates to 1 ml of the        manufacturer's bead slurry and cook for 2 hours under constant        stirring.    -   5. Following cooking transfer contents of flask to appropriate        flasks for washing.    -   6. Wash x2 in 2-propanol then resuspend in 10 ml Milli-Q water        and seal vial with parafilm.

PART A: Preparing the PVP Step Essential Recommended Avoid 1. Weigh outrequired amount of PVP (60 PVP molecules per nm² of S.A. of silicaspheres used) in 15 ml screw cap glass vial 2. Dissolve in 2 ml Mediawith Following Heating during of the 9:1 dissolved suspension ofultrasonication (9 ml) CHCl₃: (1 ml) PVP appears PVP in 1-2 ml ofExcessive 2-propanol solvent clear solvent, shake & ultrasonicationunder gentle stirring PVP is vortex over 15 min for 1 hour or shakethoroughly Prepare PVP an and vortex until dissolved hour before usemedia appears clear by dissolving under stirring 3. Recombine with stock9:1 solution

PART B: Passivating Nanocrystals to functionalized 5 μm Silica BeadsStep Essential Recommended Avoid 1. Mix 200-500 μl Shake manually for1-2 APS functionalized Not APS/MPS minutes silica beads have Shakingfunctionalized SiO₂ higher affinity for the slurry ((0.02 g/ml) &quantum dots 50-200 μl Qdots in 1.5 ml Eppendorf tube 2. Put OnMotorized At least 15 min-1Hour Allow to rotate for 1 wheel at Min-rpmMin-rpm hour 3. Add 2-propanol to Shake, Vortex & break the 2 phasescentrifuge @ 3600 rpm shake, vortex then for 10 secs centrifuge @ Checkthat pellet 3600 rpm 10 secs fluoresces under UV then discard the lightsupernatant 4. Resuspend pellet Shake vigorously and Can also use inCHCl₃, shake and ultrasonicate 2-propanol as the wash Ultrasonicate(Blitz) Check that pellet solvent then centrifuge @ fluoresces under UV3600 rpm 10 secs. light when supernatant Discard the has been discardedafter supernatant and final wash, if it does repeat wash continue ontostep 5, otherwise start again 5. Resuspend washed Resuspend the washedAdd resuspended pellet pellet in PVP pellet with a portion (1-2 ml)under-stirring solution prepared in of PVP solution PART A, and pipette2-3 times to then combine with maximize sample stock PVP solution &recovery then combine Ultrasonicate briefly with stock PVP solution thenallow to stir Use a 15 ml screw cap over night. glass-vial for reactionvessel 6. Allow to stir Seal screw-cap vial with Concave overnight. Ifsample Para-film Bottom is successful wash x2 Smooth stir motion vialsin 2-propanol then No heating cover with aluminium foil and store at 4°C.

PART C: TEOS Coating of PVP capped Nanocrystal-dopped microspheresRecom- Step Essential mended Avoid 1. Take 1-5 ml of Ultrasonicate yourPVP capped (only Blitz) microsphere sample sample 2. Combine 1:1 withstock 4.2% NH₃ solution (e.g. 1 ml of PVP capped bead then 1 ml of 4.2%NH₃ solution) 3. Under stirring Add TEOS while tip is Con- administer100 μl of within media but add to cave TEOS [5 μl pure side of vial orcentre of Bottom TEOS in 1 ml reaction media vials 2-propanol (1:200Seal screw-cap vial solution)] solution to with Para-film the NH₃/PVPFlat-Bottom screw-cap Microsphere solution vial and allow to stir Smoothstirring-motion overnight. NO HEATING! 4. Following Cover sample withsuccessful TEOS aluminum foil and store coating wash process @ 4° C. canbe performed Repeat wash at least 3x with 2-propanol i.e. add 1 ml2-propanol, shake well, ultrasonicate (Blitz), then centrifuge sample @3600 rpm for 10 s and discard supernatant and resuspend in fresh 2-propanol.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any to or more of said steps or features.

BIBLIOGRAPHY

Baird and Myszka, J Mol Recognit 14:261-268, 2001;

Casu, Ann NY Acad Sci 556:1-17, 1989;

Casu, Adv Carbohydr Chem Biochem 43:51-134, 1985;

Conrad, Heparin binding proteins Academic Press, San Diego, 1998;

Faham et al. Science 271:1116-1120, 1996;

Karlsson and Stahlberg, Anal Biochem 228:274-280, 1995;

Lander and Selleck, J Cell Biol 148(2):227-232, 2000;

Li et al. Science 299:840-843, 2003;

Lin et al. Science 278:840-843, 1997;

Lyon et al. J Biol Chem 269:11216-11223, 1994;

Maccarana et al. J Biol Chem 268(32):23898-23905, 1993;

Malmqvist, Nature 361:186-187, 1993;

Rich and Myszka, J Mol Recognit 15:352-376, 2002;

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1. A method of detecting an analyte, said method comprising contactingat least one set of microspheroidal particles with a sample putativelycomprising said analyte, wherein each particle within a set ofmicrospheroidal particles comprises an optically detectable label and animmobilized putative binding partner of said analyte wherein eachparticle set has a defined Whispering Gallery Mode (WGM) profile,wherein binding of said analyte to said immobilized binding partnerresults in a change in said WGM profile indicated by a spectral shift inthe optically detectable label of said at least one set ofmicrospheroidal particles which is indicative of the presence of saidanalyte.
 2. The method of claim 1, wherein the optically detectablelabel is a flurochrome.
 3. The method of claim 2, wherein eachmicrospheroidal set of particles is labled with a different flurochrome.4. The method of claim 1, wherein each microspheroidal set is labeledwith a different immobilized putative binding partner of said analyte.5. The method of claim 1, wherein each microspheroidal set of particlesis a different size.
 6. The method of claim 1, wherein eachmicrospheroidal set of particles have two or more of: a. a differentoptically detectable label; b. a different size; and/or c. a differentimmobilized binding partner of an analyte.
 7. The method of claim 1wherein said microspheroidal particle comprises a material selected fromthe group consisting of silica, latex, titania, tin dioxide, yttria,alumina, and other binary metal oxides, perovskites and otherpiezoelectric metal oxides, sucrose, agarose and other polymers.
 8. Themethod of claim 7, wherein said particle comprises silica.
 9. The methodof claim 1 wherein said particle is a substantially spherical orspheroidal particle.
 10. The method of claim 5 wherein said particlecomprises a diameter of about 300 nm to about 30 μm.
 11. The method ofclaim 1 wherein said optically detectable label is a molecule, atom orion which emits fluorescence.
 12. The method of claim 1 wherein saidoptically detectable label is a molecule, atom or ion which emitsphosphorescence.
 13. The method of claim 1 wherein said opticallydetectable label is a molecule, atom or ion which emits incandescence.14. The method of claim 1 wherein said optically detectable label isdetectable in any one or more of the ultraviolet, visible, near infrared(NIR) and/or infrared (IR) wavelength ranges.
 15. The method of claim 11wherein said optically detectable label is detectable in the visiblewavelength range.
 16. The method of claim 1 wherein said opticallydetectable label comprises a label selected from the group consisting ofa fluorophore, a semiconductor particle, a phosphor particle, a dopedparticle, a nanocrystal and a quantum dot.
 17. The method of claim 16wherein said optically detectable label is a fluorophore.
 18. The methodof claim 16 wherein said optically detectable label is a quantum dot.19. The method of claim 1, wherein said immobilized binding particlecomprises a nucleic acid molecule.
 20. The method of claim 19 whereinsaid nucleic acid molecule comprises DNA.
 21. The method of claim 19wherein said nucleic acid molecule comprises RNA.
 22. The method ofclaim 1, wherein said immobilized binding particle comprises a peptide,polypeptide or protein.
 23. The method of claim 22, wherein saidpeptide, polypeptide or protein is an enzyme.
 24. The method of claim22, wherein said peptide, polypeptide or protein is an antibody.
 25. Themethod of claim 1, wherein said immobilized binding particle comprises acarbohydrate molecule.
 26. The method of claim 25, wherein saidcarbohydrate is a glycosaminoglycan molecule.
 27. The method of claim 1wherein the modulation of said WGM profile comprises a red-shift of oneor more peaks in said profile.
 28. The method of claim 1 wherein themodulation of said WGM profile comprises a blue-shift of one or morepeaks in the profile.
 29. The method of claim 27 wherein the red-shiftcomprises a wavelength change of said peak or peaks of between 1 and 100nm.
 30. The method of claim 29 wherein the red-shift comprises awavelength change of said peak or peaks of between 1 and 20 nm.
 31. Themethod of claim 1 wherein the modulation of said WGM profile comprisesthe appearance of one or more peaks in one or more of said WGM profile.32. The method of claim 1 wherein the modulation of said WGM profilecomprises the disappearance of one or more peaks in one or more of saidWGM profile.
 33. The method of claim 1 wherein the WGM profile isdetermined by using a confocal microscope in conjunction with aspectrometer
 34. The method of claim 1 wherein the WGM profile isdetermined using ann array scanner in conjunction with a spectrometer.35. The method of claim 1 wherein the WGM profile is determined by adevice which measures light from individual particles in conjunctionwith a spectrometer
 36. The method of claim 35, wherein the device in aflow cytometer.
 37. An analyte detected by the method of claim
 1. 38.The method of claim 28 wherein the blue-shift comprises a wavelengthchange of said peak or peaks of between 1 and 100 nm.
 39. The method ofclaim 38 wherein the blue-shift comprises a wavelength change of saidpeak or peaks of between 1 and 20 nm.